Titolo della tesi: FGFR expression profile, keratinocyte proliferation/differentiation markers and EMT-related genes in AK: a possible predictive factor for AK malignant progression
Actinic Keratosis (AK) is the UV-induced preneoplastic skin lesion which has been clinically classified in keratinocytic intraepithelial neoplasia (KIN) of ‘low’(KIN I), ‘intermediate’ (KIN II) and ‘high’(KIN III) grade. During KIN progression, the downregulation of several keratinocyte differentiation markers has been observed (Lambert et al., 2014), while the epithelial-mesenchymal transition (EMT) phenotype it has been recently described as a typical feature of some aggressive SCCs that directly develop from KIN I through the so-called differentiated pathway. More recently, the presentation of the FGF1 and FGF2 inhibitor (Dobesilate) as a new, effective therapeutic strategy strongly suggested that the deregulation of FGF/FGFR axis could contribute to AK pathogenesis . In this regard, our recent findings underlining that the altered FGFR2 splicing and the consequent aberrant expression of the mesenchymal FGFR2c variant in epithelial context induce impairment of differentiation and EMT induction, strongly encouraged us to investigate if the FGFR2 isoform switch could also take place in AK-keratinocytes possibly contributing the rapid progression of KIN I toward aggressive SCCs.
The detailed molecular analysis performed in this work, have first highlighted that the early differentiation markers K1 and DSG1 appeared progressively downregulated during KIN progression, while the downmodulation of the terminal differentiation marker FIL was particularly marked just in some of the KIN I lesions suggesting that FIL could be one of the markers for that KIN I lesions destined to a rapid malignant progression toward SCC. In contrast, a modulation of epithelial/mesenchymal markers and an induction of the transcription factors Snail1 and ZEB1, compatible with pathological EMT, were detectable in all the analyzed AK samples, without significant correlation with KIN progression. Regarding FGFR expression, we found that a modulation of FGFR2b/FGFR2c isoforms, compatible with an FGFR2 isoform switch, as well as the upregulation of FGFR4, are detectable starting from KIN I lesions, encouraging us to suppose that they could be key events detectable starting from the early steps of AK pathogenesis. In contrast, the expression of FGFR3c was detected mainly in KIN II and KIN III lesions, suggesting that the appearance of this receptor could be a late event in AK progression. Finally, the increased expression of FGF2 mRNA in the dermal portion of all KIN lesions further supported the idea that aberrant epidermal/dermal paracrine loops based on FGF2/FGFR axis could contribute to AK pathogenesis. It is worth to note that, particularly in one of the KIN I samples a strong induction of Snail1, as well as an upregulation of FGFR2c, FGFR4, and FGF2 was observed, encouraging us to further assess if these proteins could be specific molecular markers, whose expression profile could in future help to quickly intercept KIN I lesion destined to a rapid, direct progression toward SCC.
In the second part of this workwe pointed on the identification pof the specific signaling pathways downstream FGFR2c responsible for its oncogenic outcome in epithelial context. addressing particular attention on PKCe, whose overexpression alone was sufficient to induce an EMT-related phenotype in epithelial cells. Our results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, the depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction. Therefore, the expression/activation of PKCε could represent an additional marker useful to identify that KIN I lesion destined to a rapid progression toward SCC.
Our results represent the first step to advance the understanding of the molecular bases of FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological pre-neoplastic and finally oncogenic profiles.